Biological membranes and related systems are being investigated via atomic force microscopy (AFM) and other biophysical technologies in this project containing several collaborations. (1) We continue our collaboration with Drs. B. J. Litman and S.-L. Niu (NIAAA, NIH), using atomic force microscopy (AFM) to characterize at sub-nanometer resolutions structure and function of the protein rhodopsin, a G-protein coupled receptor (GPCR), in native rod outer segment membranes of the vision pathway and in reconstituted membranes with dipalmitoylphosphatidylcholine (DPPC, di16:0PC) and polyunsatuatrated lipids such as DDPC (di-22:6n-3PC). We have obtained new AFM data on a broader range of native vision disks and reconstituted rhodopsin/membrane system over a broader set of conditions. We have developed new AFM imaging analysis methods and biophysical simulations to further reveal rhodopsin molecule organization and to explore the connection between rhodopsin signaling and the lipid membrane environment. (2) We have advanced our cutting edge AFM studies of clathrin coated vesicles (CCVs) in collaboration with Dr. Ralph Nossal (NICHD, NIH) and Drs. Eileen M. Lafer and K. Prasad (University Texas, San Antonio). CCVs are important biological membrane and multi-protein complexes that play a central role in receptor-mediated endocytosis and intracellular trafficking from the trans-Golgi network. We have measured the mechanical properties of CCVs using AFM and dedicated biophysical data analysis methods. We have confirmed the topological clathrin coat structures known from electron microscopy and moved further to yield CCV deformation as a quantitative response to the compression force in the 100 pN range. Modeling CCVs as composite elastic thin shells, we estimate that the bending rigidity of intact CCVs is about 10 times that of its constituting outer clathrin polyhedral lattice and inner phospholipids membrane. This suggests that the adaptor protein layer in native CCVs provides a partial coupling between its clathrin coat and the inner lipid membrane loaded with receptors and ligands. (3) We continue our biological membrane and related studies using AFM, biophysical data analyses, nanotechnology, and other scientific methods.